General Introduction
Molecular diagnostics and other DNA based approaches have gained increasing focus in sectors such as Discovery, R&D, and in various branches of diagnostics. However, whatever target is being analysed by assays or screening, all DNA based approaches face the same challenge, namely to generate sufficient amounts of available target relative to background DNA.
By increasing the presence of low abundance target DNA, at the expense of undesirable background DNA in a complex mixed DNA sample, traditional molecular methods, such as clone library construction, natural product discovery, PCR diagnostics, hybridizations, sequencing, metagenomics, and a variety of other molecular approaches are enabled.
The following describes methods currently used and the challenges of low abundance, mixed DNA samples.
PCR Diagnostics
In recent years, PCR tests have been extensively developed for routine diagnostics of infectious diseases in clinical microbiology. PCR is suited for rapid detection of bacteria directly in clinical specimens, allowing early, sensitive and specific laboratory confirmation of related diseases [1]. Moreover, it allows a rapid assessment of the presence of antibiotic resistance genes or gene mutations. An approach combining pathogen detection, their mechanisms of antibiotic resistance, their virulence factors and bacterial load in clinical samples could lead to profound changes in the care of these infected patients. Therefore, complex and multiplex PCR assays are currently being developed to enhance the field of molecular diagnostics.
However, PCR based diagnoses from mixed samples are often associated with challenges, as the presence of PCR products can arise from more than one DNA source in mixed complex samples. The presence of a specific antibiotic gene combined with the presence of specific bacterial strain does not necessarily mean that a resistant bacterial strain was present in the original sample. It only indicates that both a resistance gene and the bacterial strain were present in the sample, not necessarily that they arise from the same cell. Approaches to circumvent this problem have been suggested where the integration site of the antibiotic resistance gene was targeted by specific primers also known to target a specific bacterial strain. However, the exact integration site will then need to be known and this limits the use of the method.
DNA Sequencing
Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous applied fields such as diagnostic, biotechnology, forensic biology, and biological systematics. The rapid speed of sequencing and decreasing cost attained with modern DNA sequencing technology has been instrumental in the sequencing of DNA sequences and the total amount of DNA sequenced worldwide is increasing rapidly.
While sequencing of pure samples is now standard procedure, sequencing of mixed samples of DNA is still challenging, costly and time consuming. When the target fragment is present at low frequency in a mixed nucleotide sample, for instance in a swab, faeces or blood sample, one must first make a clone or fragment library and then either sequence the complete library, or make smaller PCR fragments and sequence these. Sequencing of the complete library is expensive and time consuming and sequencing of PCR fragments is only possible if the sequence is already known to a very large extent and will return relatively short fragments which cannot be assigned to the same molecule or organism. If only part of the target sequence is known, PCR will not be possible and metagenome sequencing is needed.
There is therefore a need for a method for increasing the frequency of rare nucleotide molecules in a mixed sample of nucleotide molecules in order to decrease the cost and time of sequencing mixed samples.
Metagenomics
Metagenomics refers to a general sequencing approach, where a complex mixture of DNA is differentiated into small fractions and sequenced individually. The system does not rely on culturability and is, thus, applicable for both samples that can and cannot be cultivated in a laboratory.
Although metagenomics can be applied in some cases to describe complex samples, it is frequently the case that researchers prefer a specific subset of the genome or mixture of genomes, rather than the entire sequence of a sample or mixed sample of genomes [2]. Thus, there exists a strong need for flexible targeting methods that are matched to the individual requirements of the various techniques and studies. Although such techniques do exist they are often based on hybridization assays, and have the prerequisite of extensive sequence knowledge or have low sensitivity.
Therefore, a strong demand is present, for specific enrichment of a pre-defined sub-fraction of a complex mixture, if metagenomics is to be applied for analyzing rare/low abundance DNA fragments or targets.
Discovery
Different industries have different motivations to explore the vast resource that lies within uncultivated microbial diversity. Currently, white (industrial) biotechnology seems to play a central role in the establishment of the sustainable modern society. It is a commonly accepted assumption that only a small sub-fraction of the natural microbial biodiversity is available for screening. In 1990, Torsvijk et al. [3] estimated that at best only 1% of the naturally occurring microbial diversity of a soil sample could be cultivated under laboratory conditions. Hence, a huge potential lies within the discovery of yet unknown natural products, and also in biotechnological techniques that enable access to the unknown majority of DNA present in natural and environmental samples. Unfortunately, enormous amounts of sequencing is required if the otherwise unavailable information is to be retrieved.
Targeted enrichment of DNA fragments encoding industrially relevant proteins or enzymes would substantially decrease the required amount of sequencing.